Recently, hepatitis C virus (HCV) strains that are infectious in human hepatoma cells were identified. This provides the first opportunity to study how the virus enters and exits liver cells. We propose a combinatorial analysis to dissect the roles of cellular membrane trafficking pathways in HCV infection. We will integrate RNA interference (RNAi) analysis, live cell microscopy, viral genetics, and biochemistry to probe viral trafficking in the infected cell. This will be especially fruitful, since HCV uses membrane trafficking pathways at each step of its life cycle in a series of complex interactions. An siRNA library was assembled that targets 144 genes that participate in all known membrane trafficking pathways. We have interrogated this library and identified 48 host genes that are required for HCV infection. These genes cluster into specific pathways of clathrin-mediated endocytosis, cytoskeletal re- organization, endocytic trafficking, phosphatidyl inositol (PI) signaling, membrane re-organization, Golgi structure, and components of the secretory pathway. We have assigned roles for these genes in viral infection using HCV cell culture systems that isolate specific stages of the viral life cycle, including HCV pseudo-particles (HCV entry) and sub-genomic HCV replicons (HCV replication). HCV trafficking in the infected cell will then be analyzed by combining the specific inhibitory siRNAs with microscopy and biochemical approaches to determine the sequence of events in HCV entry and egress. Genetic and biochemical approaches are described to define the functions of endocytic trafficking and phosphatidyl inositol (PI) signaling in the formation of HCV replication complexes. The specific aims are: 1. Molecular characterization of membrane trafficking pathways for HCV entry into cells. We will define the requirements for 17 host genes in 3 pathways for the distinct stages of HCV entry: 2. Identify the mechanism of HCV-induced membrane re-organization for establishing replication. We will investigate the function of endocytic trafficking and PI-4 kinase IIIa in membrane re-arrangements leading to the establishment of HCV replication complexes. 3. Identify membrane trafficking pathways important for HCV particle maturation and egress. We will target the host genes involved in egress to characterize the stages of virion egress and maturation. These studies will illuminate major stages of the viral life cycle that are currently poorly characterized. Additionally, these data will further develop our understanding into the components and regulation of human membrane trafficking pathways using a highly relevant and complex probe (HCV).